Chlorophyll is a light-absorbing pigment found in all green plants, as well as algae and some bacteria. It is essential for photosynthesis, a process where light energy from the sun is converted to stored chemical energy. When chlorophyll absorbs sunlight it re-emits a small fraction of this light as fluorescence, and variations in the proportion of absorbed energy that is re-emitted can be related to the efficiency of the photosynthetic system. By examining subtle changes in qualities of chlorophyll fluorescence (for example variable fluorescence intensity) in response to specific experimental conditions it can be determined whether photosynthesis is proceeding optimally or whether it has been adversely affected by the environment. In order for the fluorescence signal to be clearly distinguished from background light when measurements are made with ambient light present, the signal is stimulated by a brief typically modulated “excitation” light (typically of order of milliseconds), and the returned fluorescence “emission” signal is read immediately afterwards. Additional lights are often applied to the sample in order to alter its physiological state, thereby extracting more information pertaining to the condition of the sample.
Based on these general principles, numerous chlorophyll fluorescence techniques have been developed and used by many research groups since the mid 20th century. These techniques provide information useful in describing the condition of the photosynthetic organism, are used widely in plant and agricultural research and are increasingly used in the commercial sector.
A commonly used technique for directing a beam of excitation light to a sample, and receiving the emission signal from that sample is to use a beam-splitting apparatus, where either the excitation or emission light is directed through a partially mirrored material positioned at 45 degrees to the beam direction. The mirrored material used is generally designed to either allow transmission of the excitation beam and to reflect the emission beam or allow reflection of the excitation beam and transmission of the emission beam. This beam-splitting optical arrangement allows the emission source and the detector to be positioned separately yet in the direct path of the emitted or received beam. While effective, this system requires a relatively precise and stable spatial optical arrangement and a high-quality mirror for it to be effective. This adds to the complexity, cost and physical size of the apparatus.
Within the suite of fundamental fluorescence measurements that can be obtained from modulated fluorometers, the variable fluorescence of temporarily dark-acclimated plant material is generally, but not exclusively, described by the parameter Fv/Fm which remains a vital source of information that describes the photosynthetic system of the plant material measured when it is in a state of partial or complete relaxation, The parameter Fv/Fm is a measure of the efficiency with which light absorbed by the photosynthetic material drives photochemistry; Fv is a measure of the variable fluorescence; and Fm is a measure of maximum fluorescence. Reference is made to publications by Schreiber, U & Falkowski, P and references cited thereby for more detail regarding these measurements.
The parameter Fv/Fm obtained from a dark acclimated sample can then be compared with variable fluorescence values obtained from the photosynthetic system when it is acclimated to ambient light. The protocol for obtaining the Fv/Fm measurement generally requires the sample to be kept in the dark for an interval of at least 10 to 15 minutes prior to taking the measurement. It is generally unnecessary to dark-acclimate the entire plant; rather a small portion of the plant is dark-acclimated (i.e. “sample”). The term “dark-acclimation” is used here to describe the deliberate exclusion of substantially all ambient light from reaching the sample. This includes the exclusion of most or all natural ambient light (e.g. sunlight), artificial ambient light (e.g. overhead lights in a room or enclosure), or any artificial light treatment.
In addition to measuring fluorescence emitted from a photosynthetic sample, useful information pertaining to the physiological quality of the sample can be obtained by measuring the absorbance and reflectance of light; these measurements may also be best made in the dark so that ambient light does not interfere with the sample or measurement. These measurements can also be usefully performed on non-living samples for e.g. colour measurement, and are generally performed using spectrophotometric and colourimetric techniques, although spectrofluorometry has also been used.
The ability to exclude unwanted ambient light from reaching a sample is also necessary when making measurements that require the sample to be exposed to artificial light treatments. By deliberately excluding unwanted ambient light when making these fluorescence measurements one can be certain that the sample is only responding to the artificial light provided. As for dark-acclimation, these measurements require an operator to manually cause the exclusion of external light.
While obtaining measurements from dark-acclimated samples (or from samples where ambient light is excluded and artificial light treatments are applied) is required, equally important is the ability to ensure the sample is exposed to ambient irradiance when not taking measurements or excluding ambient light. Thus the operator needs to be able to ensure the sample is unshaded for some interval of time.
Known methods for temporarily placing a sample in darkness or temporarily excluding the influence of ambient light when conducting fluorescence measurements (and other optical measurements including absorbance transmittance and reflectance) are either to manually exclude all light when conducting the measurement by applying an opaque cover, or when using artificial lights for ambient illumination, simply turn off the lights either manually or automatically when taking the measurements.
The disadvantages inherent in the above methods are that an operator is required to be present whenever the ambient light is to be excluded. This is required because the operator must physically apply opaque covers to the sample to prevent ambient light from reaching the sample, and then remove the cover when the sample is again to be exposed to ambient light. The farmer method can present significant limitations in situations where it is undesirable to have an operator attending the sample for long periods. For example, it would be undesirable for the operator to be required to make regular and frequent measurements over a 24 hour period when the sample is underwater or in a very cold and wet environment. The difficulty in ensuring operator safety under such a sampling regime can be significant and deter the making of any measurements. One disadvantage of the latter method, (i.e. turning off artificial lights), is that this may dark-acclimate the entire sample, and this is generally not desirable.